In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons, and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene;it is an idiosyncratic complication of some medical drug use;it occurs as a rare event in pregnancy and following seronegative hepatitis;and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. Our randomized control trial of horse versus rabbit antithymocyte globulin (ATG) has been completed and published. One hundred and twenty patients were randomized between these two biological agents, which have been utilized interchangeably in the clinic. We found a highly significant difference in the hematologic response at six months in favor of horse ATG, after which 68% of patients improved compared to a 37% response rate for rabbit ATG. Survival at three years was 96% for patients in the horse ATG arm as compared to 76% for patients treated with rabbit ATG, also stastically significant. Correlated with the clinical differences were distinct patterns of immunologic reconstitution after lymphocyte depletion. Rabbit ATG was more lymphodepleting, and lymphocyte numbers remained low for a longer period. CD8 cells were comparably decreased by both ATGs, but rabbit ATG administration led to a more profound decline in CD4 cells, including CD4/CD25 T regulatory cells. The clinical results of this study have immediate impact on the treatment of aplastic anemia patients outside of the United States, as in Europe, Asia, and much of South America horse ATG is not available. Further investigation in the laboratory of the effects of ATGs are underway; these studies have focused on various aspects of ATG treatment. In a continuation of studies of the immune system after ATG, we have undertaken analysis by microarray of gene expression of CD4 cells as they recover. We have also examined ATG kinetics, finding that both horse and rabbit ATG remain free and active for binding in the plasma of patients for weeks to months after initial administration. In studies of serum sickness, we have noted marked differences in various plasma cytokines as a result of immune complex formation. Many of these laboratory studies are in the context of global approaches to the immune system using multiplex PCR for plasma cytokines and deep lymphocyte phenotyping for very large numbers of mononuclear cell subsets. Such an approach has yielded unexpected and interesting data. We can distinguish between hypercellular MDS and aplastic anemia based on plasma cytokine patterns. Because thrombocytopenia is a common feature of both syndromes, we have also inferred from the unbiased data generated by these approaches potential hypotheses concerning normal physiology and pathophysiology. When we compared the patterns of cytokines in thrombocytopenia due to bone marrow failure (as in aplastic anemia and MDS) and in idiopathic thrombocytopenia purpura (ITP), distinct cytokines were associated with both (CD40L, CXCL5, CCL5, and EGF ). In vitro and in animal model, we confirmed that these cytokines were platelet and megakaryocyte in origin. In other clinical studies, we have completed most of our Campath clinical trials directed at refractory and relapsed aplastic anemia. In patients with untreated severe aplastic anemia, Campath was the third arm of our randomized trial and we abandoned due to lack of efficacy: only occasional patients appeared to respond to Campath alone as primary immunosuppression. In refractory aplastic anemia, CAMPATH appeared equivalent to rabbit ATG but can be administered without cyclosporine, yielding response rates of 30-40% hematologic improvement. In relapsed aplastic anemia, a majority of patients will respond to a course of Campath, achieving transfusion-independence. Finally, we have tested whether prolonged cyclosporine administration for more than six months after initial ATG impacted the rate of relapse;European studies have suggested the utility of a two year regimen. However, in our patients, there was no significant difference in relapse nor in malignant evolution in patients receiving cyclosporine in tapering doses over two years. To address the variability of responses between horse and because rabbit ATG and rabbit ATG, despite being the inferior agent, is the only one available in much of the world, we have initiated several protocols to assess the utility of cyclophosphamide, in combination with cyclosporine, in patients with severe aplastic anemia and cyclophosphamide, in combination with fludarabine, in patients with refractory disease. Cyclophosphamide is a defined chemical with a known ability to induce responses in bone marrow failure, has advantages of cost and availability, compared to ATG. However, our previous experience with high dose cyclophosphamide was of significant toxicity, especially prolonged nutropenia and susceptibility to fatal fungal infections. In our new trials, we will take advantage of the availability of antifungal drugs that can be administered prophylactically or therapeutically. To date, 19 patients have entered our protocol for severe aplastic anemia on presentation and two patients have been entered to the protocol for refractory aplastic anemia.